Use of cannabinoids in the treatment of Angelman syndrome

ABSTRACT

The present invention relates to the use of cannabidiol (CBD) in the treatment of Angelman syndrome (AS). CBD has been shown to be particularly effective in improving anxiety in rodent models of AS. The CBD is preferably substantially pure. It may take the form of a highly purified extract of Cannabis such that the CBD is present at greater than 98% of the total extract (w/w) and the other components of the extract are characterised. Alternatively, the CBD is synthetically produced. Alternatively, the CBD may be used as a botanical drug substance (BDS) from a Cannabis plant in which CBD is the predominant cannabinoid. The CBD may also be present in combination with other cannabinoids and non-cannabinoid components such as terpenes. In use the CBD may be used concomitantly with one or more other medicaments. Alternatively, the CBD may be formulated for administration separately, sequentially or simultaneously with one or more medicaments or the combination may be provided in a single dosage form. Where the CBD is formulated for administration separately, sequentially or simultaneously it may be provided as a kit or together with instructions to administer the one or more components in the manner indicated. It may also be used as the sole medication, i.e. as a monotherapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Entry under 35 U.S.C. 371 ofPCT/GB2017/053735, filed Dec. 13, 2017, which claims priority toGB1621480.1, filed Dec. 16, 2016.

FIELD OF THE INVENTION

The present invention relates to the use of cannabidiol (CBD) in thetreatment of Angelman syndrome (AS).

The CBD is preferably substantially pure. It may take the form of ahighly purified extract of Cannabis such that the CBD is present atgreater than 98% of the total extract (w/w) and the other components ofthe extract are characterised. Alternatively, the CBD is syntheticallyproduced.

In a further alternative the CBD may be used as a botanical drugsubstance (BDS) from a Cannabis plant in which CBD is the predominantcannabinoid. In such an embodiment the CBD may be present in combinationwith other cannabinoids and non-cannabinoid components such as terpenes.

In use the CBD may be used concomitantly with one or more othermedicaments. Alternatively the CBD may be formulated for administrationseparately, sequentially or simultaneously with one or more medicamentsor the combination may be provided in a single dosage form. Where theCBD is formulated for administration separately, sequentially orsimultaneously it may be provided as a kit or together with instructionsto administer the one or more components in the manner indicated. It mayalso be used as the sole medication, i.e. as a monotherapy.

BACKGROUND TO THE INVENTION

Angelman syndrome (AS) occurs in approximately 1 in 12,000 to 15,000individuals and is caused by abnormalities on chromosome 15. Individualswith AS typically show severe to profound learning disability,significant difficulties with mobility and communication in addition toseizures.

It has been suggested that between 50% and 80% of individuals with ASmeet the criteria for autism spectrum disorder.

Typical characteristics of Angelman syndrome include: delayeddevelopment which is usually noticeable from 6-12 months of age; severelanguage impairment with little or no speech; movement and balanceproblems (ataxia); frequent seizures (epilepsy) in around 85% of cases;a small head size (microcephaly); sociable behaviour with frequentsmiling.

A genetic anomaly responsible for AS occurs by chance around the time ofconception. The UBE3A gene is either absent or malfunctions. A childusually inherits one copy of the UBE3A gene from each parent. Bothcopies are switched on (active) in most of the body's tissues. However,in certain areas of the brain, only the gene inherited from the motheris active. In most cases of AS (about 70%), the child's maternal copy ofthe UBE3A gene is missing, which means there's no active copy of theUBE3A gene in the child's brain.

Testing compounds for their effectiveness on signs and symptoms ofAngelman syndrome is challenging given that this disorder has so manydifferent affected symptom domains.

The UBE3A mouse model is used to evaluate a compounds effectiveness inthe treatment of AS. This model has been shown to recapitulate many ofthe phenotypic features of AS, including motor dysfunction, increasedseizure susceptibility, and hippocampal-dependent learning and memorydeficits in mice with the knockout gene.

The NOR test is used to evaluate cognition, particularly recognitionmemory, in rodent models of CNS disorders, such as Angelman syndrome.The test is based on the tendency of rodents to spend more timeexploring a novel object than a familiar one. The choice to explore thenovel object reflects the use of learning and recognition memory.

The NOR test is conducted in an open field arena with two differentkinds of objects. Both objects are generally consistent in height andvolume, but are different in shape and appearance. During habituation,the animals are allowed to explore an empty arena. Twenty-four hoursafter habituation, the animals are exposed to the familiar arena withtwo identical objects placed at an equal distance. The next day, themice are allowed to explore the open field in the presence of thefamiliar object and a novel object to test short-term and long-termrecognition memory. The time spent exploring each object and thediscrimination index percentage is recorded. This test is useful forassessing cognitive dysfunction in rodent models of AS.

There is no specific therapy for Angelman syndrome. Medication isusually required to control seizures. Physical and occupationaltherapies, communication therapy, and behavioral therapies are alsoimportant.

The endocannabinoid system has been linked to physiological progressionof autism spectrum disorders, possibly implicating CB1 and CB2receptors.

The phytocannabinoids are known to interact with the endocannabinoidsystem.

The phytocannabinoid tetrahydrocannabinol (THC) in the form ofdronabinol, CB1 agonist, has been used to treat an autistic child (Kurzand Blass, 2010). Problems associated with the use of CB1 agonists arepsychoactivity, anxiety and hallucinations.

Patent application WO 2014/146699 describes the use of CB1 receptorantagonists in the treatment of diseases associated with dendriticabnormalities. Such diseases include AS and RS. The application isexemplified by the use of rimonabant in the FMR1 knockout mouse modelwhich is a model of FXS.

The CB1 antagonist, rimonabant, has been shown to have serious sideeffects which limit its use, such as suicide ideation.

The present application relates to the use of a phytocannabinoid whichis neither a CB1 agonist nor antagonist. CBD is known to have a lowaffinity for the CB1 receptor (Mechoulam et al. 2007).

To date there are no studies of the use of CBD in the treatment of AS.Such symptoms as described above are difficult to treat, therefore manypatients with AS have unmet needs with respect to the treatment of theirdisease.

BRIEF SUMMARY OF THE DISCLOSURE

In accordance with a first aspect of the present invention there isprovided Cannabidiol (CBD) for use in the treatment of Angelmansyndrome.

In this aspect, treatment of Angelman syndrome encompasses the treatmentof the condition as a whole, as opposed to the individual symptoms.

In accordance with a second aspect of the present invention there isprovided Cannabidiol (CBD) for use in the treatment of one or moresymptoms or characteristics associated with of Angelman syndrome.

Preferably the symptoms or characteristics associated with Angelmansyndrome are one or more of: movement disorders; anxiety and/orcognitive dysfunction.

In accordance with a third aspect of the present invention there isprovided Cannabidiol (CBD) for use in the treatment of movementdisorders associated with Angelman syndrome.

In accordance with a forth aspect of the present invention there isprovided Cannabidiol (CBD) for use in the treatment of anxietyassociated with Angelman syndrome.

In accordance with a fifth aspect of the present invention there isprovided Cannabidiol (CBD) for use in the treatment of cognitivedysfunction associated with Angelman syndrome.

In a further embodiment the CBD is for use in combination with one ormore concomitant medicaments which may be taken by the patient to treatthe condition and/or one or more symptoms associated therewith. Such as,for example, melatonin for sleeping problems, SSRI for depression,anticonvulsants for epilepsy, methylphenidate for ADHD or antipsychoticsfor aggression or self-harming behaviour. Preferably the one or moreconcomitant medicaments is an anti-epileptic drug (AED).

In a preferred embodiment the CBD is substantially pure. The CBD may bepresent as a highly purified extract of Cannabis which comprises atleast 98% (w/w) CBD. Preferably the extract comprises less than 0.15%THC.

In an alternative embodiment the CBD is present as a synthetic compound.

In yet a further embodiment the CBD may be used as a botanical drugsubstance, as an extract from a Cannabis plant in which CBD is thepredominant cannabinoid. The CBD may also be present in combination withother cannabinoids and non-cannabinoid components such as terpenes.

Determining an effective dose in humans will depend on, for example themode of delivery (e.g. i.v. or oral), the formulation and thebioavailability of the CBD when delivered and might range between 0.1and 100 mg/kg/day. Furthermore the fact that cannabinoids often showbell-shaped dose response curves makes determining a dose of CBD moredifficult.

Preferably the dose of CBD is greater than 0.1 mg/kg/day. Thus, for a 15kg patient a dose of greater than 1.5 mg of CBD per day would beprovided. Doses greater than 1 mg/kg/day such as greater than5/mg/kg/day, greater than 10 mg/kg/day, greater than 15 mg/kg/day andgreater than 20 mg/kg/day are also envisaged to be effective.

Preferably the CBD is provided over an extended period; more preferablythis period is at least seven days.

In a further embodiment the CBD may be used as a dietary supplement orfood additive in order to improve symptoms in Angelman syndrome.

In accordance with a sixth aspect of the present invention there isprovided a method of treating Angelman syndrome, comprisingadministering an effective amount of cannabidiol (CBD) to the subject inneed thereof.

Preferably the subject is a human.

Preferably the symptoms or characteristics associated with Angelmansyndrome are one or more of: movement disorders; anxiety and/orcognitive dysfunction.

In accordance with a seventh aspect of the present invention there isprovided a method of treating a movement disorder in an Angelmansyndrome subject comprising administering an effective amount ofcannabidiol (CBD) to the subject in need thereof.

Preferably the subject is a human.

In accordance with a eighth aspect of the present invention there isprovided a method of treating anxiety in an Angelman syndrome subjectcomprising administering an effective amount of cannabidiol (CBD) to thesubject in need thereof.

Preferably the subject is a human.

In accordance with a ninth aspect of the present invention there isprovided a method of treating a cognitive disfunction in an Angelmansyndrome subject comprising administering an effective amount ofcannabidiol (CBD) to the subject in need thereof.

Preferably the subject is a human.

The human equivalent dose (HED) can be estimated using the followingformula:

${HED} = {{Animal}\mspace{14mu}{dose}\mspace{14mu}\left( {{mg}\text{/}{kg}} \right)\mspace{14mu}{multiplied}\mspace{14mu}{by}\mspace{14mu}\frac{{Animal}\mspace{14mu} K_{m}}{{Human}\mspace{14mu} K_{m}}}$The K_(m) for a mouse is 3, for a rat the K_(m) is 6 and the K_(m) for ahuman is 37.

Furthermore when animal data is obtained using an intraperitoneal formof dosing, but the drug is to be administered using a different deliverymethod, then the HED calculated may need to be adjusted to take accountof the different mode of delivery. In the case of an oral dose the HEDmay need to be increased in order to obtain an equivalent oral dose,depending on the formulation used, mode of delivery and bioavailabilityof the active. This is something a skilled pharmacologist willrecognise.

Definitions

Definitions of some of the terms used to describe the invention aredetailed below:

The cannabinoids described in the present application are listed belowalong with their standard abbreviations.

TABLE 1 Cannabinoids and their abbreviations CBD Cannabidiol

THC Tetrahydro- cannabinol

The table above is not exhaustive and merely details the cannabinoidswhich are identified in the present application for reference. So farover 60 different cannabinoids have been identified and thesecannabinoids can be split into different groups as follows:Phytocannabinoids; Endocannabinoids and Synthetic cannabinoids (whichmay be novel cannabinoids or synthetically produced phytocannabinoids orendocannabinoids).

“Phytocannabinoids” are cannabinoids that originate from nature and canbe found in the Cannabis plant. The phytocannabinoids can be isolatedfrom plants to produce a highly purified extract or can be reproducedsynthetically.

“Substantially pure CBD” is defined as CBD that is greater than 98%(w/w) pure. More preferably greater than 98.5% (w/w) through 99% (w/w)to 99.5% (w/w) and greater.

“Highly purified cannabinoid extracts” are defined as cannabinoids thathave been extracted from the Cannabis plant and purified to the extentthat other cannabinoids and non-cannabinoid components that areco-extracted with the cannabinoids have been substantially removed, suchthat the highly purified cannabinoid is greater than or equal to 98%(w/w) pure.

“Botanical drug substance” or “(BDS)” is defined in the Guidance forIndustry Botanical Drug Products Draft Guidance, August 2000, USDepartment of Health and Human Services, Food and Drug AdministrationCentre for Drug Evaluation and Research as: “A drug derived from one ormore plants, algae, or microscopic fungi. It is prepared from botanicalraw materials by one or more of the following processes: pulverisation,decoction, expression, aqueous extraction, ethanolic extraction or othersimilar processes.” A botanical drug substance does not include a highlypurified or chemically modified substance derived from natural sources.Thus, in the case of Cannabis, BDS derived from Cannabis plants do notinclude highly purified Pharmacopoeial grade cannabinoids. In a BDScomprising cannabinoids the cannabinoid will be present in an amount ofless than 95% (w/w).

“Synthetic cannabinoids” are compounds that have a cannabinoid orcannabinoid-like structure and are manufactured using chemical meansrather than by the plant.

Phytocannabinoids can be obtained as either the neutral (decarboxylatedform) or the carboxylic acid form depending on the method used toextract the cannabinoids. For example it is known that heating thecarboxylic acid form will cause most of the carboxylic acid form todecarboxylate into the neutral form.

“Cognitive dysfunction” is defined as the loss of intellectual functionssuch as thinking, remembering, and reasoning with sufficient severity tointerfere with daily functioning. Patients with cognitive dysfunctionhave trouble with verbal recall, basic arithmetic, and concentration.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter withreference to the accompanying drawings, in which

FIG. 1 shows the effect of CBD on clasping duration in a mouse model ofAngelman syndrome;

FIG. 2 shows the effect of CBD in the rotarod test in a mouse model ofAngelman syndrome;

FIG. 3 shows the effect of CBD in the novel object recognition test in amouse model of Angelman syndrome; and

FIG. 4 shows the effect of CBD in the tail suspension test in a mousemodel of Angelman syndrome.

Data are expressed as mean±S.E.M. *p<0.05, ***p<0.001 vs WT-vehicle;^(∘∘)p<0.01, ^(∘∘∘)p<0.001 vs KO-vehicle. Two-way ANOVA followed byBonferroni post hoc test.

DETAILED DESCRIPTION Example 1: Use of Cannabidiol (CBD) in a MouseModel of Angelman Syndrome

The effect of CBD was tested in the neurological, behavioural and motordisorders in a transgenic mouse model of Angelman Syndrome.

Materials and Methods

Animals

Heterozygous mice with maternal deficiency of Ube3A (Ube3am−/p+) andwild type (Ube3am+/p+) were purchased from The Jackson Laboratory(Jackson code: B6.12957-Ube3a tm1Alb/J) and maintained in a C57BL/6background.

Animals were housed under controlled illumination (12:12-hour light/darkcycle; light on 6 hours) and environmental conditions (room temperature:20° C.-22° C., humidity: 55%-60%) with food and tap water were availablead libitum.

Drugs and Treatment

Drugs were dissolved in 1:1:18 ethanol:cremophor:0.9% saline, forintraperitoneal (i.p.) administration. Drug treatment was performeddaily for 35 days. CBD was administered at 20 mg/kg.

Behavioural Tests

Rotarod: The rotarod test assesses balance and motor coordination ofmice. Mice have been measured for the time (in seconds) of equilibriumbefore falling on a rotary cylinder by a magnet that, activated from thefall of the mouse on the plate, allows to record the time of permanenceon the cylinder. After a period of adaptation of 30 s, the spin speedgradually increased from 3 to 30 rpm for a maximum time of 5 min. Theanimals were analysed by 2 separate tests at 1-h interval in the sameday.

Clasping: The clasping test assesses ataxia in mice. Mice were suspendedby the base of the tail and their behaviours were recorded for 30seconds. The time for which the mice clasped their hind limbs wasrecorded. The time was then scored as follows: 4, 15-30 s, 3, 10-15 s,2, 5-10 s, 1, 0-5 s and 0, 0 s

Tail suspension: The tail suspension test assesses depressive-likebehaviour in mice. Mice were individually suspended by the tail on ahorizontal bar (50 cm from floor) using adhesive tape placedapproximately 4 cm from the tip of the tail. The duration of immobilitywas recorded in seconds over a period of 6 minutes by a time recorder.Immobility time was defined as the absence of escape-oriented behaviour.

Novel Object Recognition: The novel object recognition assessesrecognition memory in mice. The experiment started with the habituationperiod, during which mice were allowed to freely explore for 1 hour theapparatus which consists of a rectangular open box (40×30×30 cmwidth×length×height) made of grey polyvinyl chloride (PVC) illuminatedby a dim light. The day after each mouse was allowed to explore twoidentical objects positioned in the back left and right corners for 5min (acquisition). A video camera recorded the time spent on explorationof each object. In the test trial, which was carried out for 2 hrs afterthe acquisition, one of the two objects was replaced with a newdifferent object. The time spent exploring the object was the time thatthe mouse spent with its nose directed and within 1 cm from the object.The behaviour of mice was analyzed by an observer blind to thetreatment. Data are expressed as percentage of recognition index (RI %),which was calculated as the percentage of the time spent exploring thenovel object/time spent exploring the novel object+time spent exploringthe familiar object×100.

Statistical Analysis: Behavioural data are represented as means±SEM andstatistical analysis of these data was performed by two way analysis ofvariance (ANOVA) for repeated measured followed by the StudentNewman-Keuls for multiple comparisons to determine statisticalsignificance between different treated groups of mouse. p<0.05 wasconsidered statistically significant.

Results

FIG. 1 shows that AS mice treated with vehicle showed significantlylonger clasping duration at 10 weeks of age compared to WT mice treatedwith vehicle. In AS mice chronic treatment (30 days) with CBDsignificantly reduced clasping duration at 10 weeks of age compared toAS mice treated with vehicle.

FIG. 2 demonstrates that AS mice treated with vehicle showed asignificant motor impairment at 10 weeks of compared to WT mice treatedwith vehicle. In AS mice, chronic treatment (30 days) with CBDsignificantly reduced latency to fall compared to AS mice treated withvehicle.

Mice were assessed at the age of 7-8 weeks in the novel objectrecognition test. AS mice treated with vehicle showed a significantdecrease in the discrimination index compared with WT mice that receivedthe same treatment. FIG. 3 shows that AS mice treated with CBD slightlyincreased the discrimination index compared to AS mice treated withvehicle.

FIG. 4 shows that in the tail suspension test the time of immobilitywere significantly higher in AS mice that received vehicle compared toWT mice that received the same treatment. In AS mice treatment with CBDsignificantly reduced the duration of immobility time compared to ASmice treated with vehicle.

Conclusion

These data demonstrate that the treatment of 20 mg/kg CBD to mice whichwere deficient in the Ube3A gene and subsequently suffered symptoms toindividuals with AS, were able to reverse these symptoms. In particularCBD was able to statistically significantly improve movement and balanceproblems (ataxia) and anxiety. As such CBD is considered to be a viabletreatment option for AS.

As is shown in Example 1 above, the use of CBD in a model of Angelmansyndrome is able to produce statistically significant reversal of thesymptoms associated with this disorder.

In particular CBD has been shown to produce positive results in the TailSuspension test in this model and as such demonstrates unequivocallythat this phytocannabinoid could reverse anxiety in this disorder.

In addition, other tests in this model provide support that CBD could beused to treat additional symptoms associated with this disorder as itwas able to reduce ataxia and balance and motor coordination symptoms ina model of Angelman syndrome. As such CBD provides a real treatmentoption to individuals suffering from AS.

REFERENCES

-   Hill T et al. (2012) Br J Pharmacol. December; 167(8):1629-42.    Cannabidivarin is anticonvulsant in mouse and rat.-   Erica Zamberletti, Sarah Beggiato, Luca Steardo Jr., Pamela Prini,    Tiziana Antonelli, Luca Ferraro, Tiziana Rubino, Daniela Parolaro.    Neurobiology of Disease (2014), Volume 63, Pages 35-47. “Alterations    of prefrontal cortex GABAergic transmission in the complex    psychotic-like phenotype induced by adolescent    delta-9-tetrahydrocannabinol exposure in rats.”-   Kurz and Blass 2010 Use of dronabinol (delta-9-THC) in autism: A    prospective single-case study with an early infantile autistic    child. Cannabinoids, 5 (4) 4-6.-   Marta Kruk-Slomka, Agnieszka Michalak, Barbara Budzyn'ska, Graz'yna    Biala. Pharmacological Reports 66 (2014), 638-646. “A comparison of    mecamylamine and bupropion effects on memory-related responses    induced by nicotine and scopolamine in the novel object recognition    test in mice.”

The invention claimed is:
 1. A method for treating anxiety associatedwith Angelman syndrome, comprising orally administering to a subject inneed thereof a therapeutically effective amount of a cannabidiol (CBD)drug substance, wherein the CBD drug substance comprises at least 98%(w/w) CBD and the CBD is administered at a dose of 20 mg/kg/day.
 2. Themethod of claim 1, wherein the CBD drug substance is administered incombination with one or more concomitant medications.
 3. The method ofclaim 1, wherein the CBD drug substance is administered in combinationwith at least one anti-epileptic drug (AED).
 4. The method of claim 1,wherein the CBD drug substance is an extract of Cannabis.
 5. The methodof claim 4, wherein the extract comprises less than 0.15% THC.
 6. Themethod of claim 1, wherein the CBD is a synthetic compound.
 7. Themethod of claim 1, further comprising treating a movement disorder. 8.The method of claim 1, further comprising treating cognitivedysfunction.
 9. A method for treating anxiety associated with Angelmansyndrome, comprising orally administering to a subject in need thereof atherapeutically effective amount of cannabidiol (CBD), wherein: the CBDis an extract of Cannabis which comprises at least 98% (w/w) CBD andless than 0.15% THC; and the CBD is administered at a dose of 20mg/kg/day.
 10. The method of claim 1, wherein the CBD drug substancecomprises greater than 98.5% (w/w) CBD.
 11. The method of claim 10,wherein the CBD is an extract of Cannabis.
 12. The method of claim 1,further comprising treating seizures.
 13. The method of claim 10,wherein the CBD is a synthetic compound.
 14. The method of claim 1,wherein the CBD drug substance comprises greater than 99% (w/w) CBD. 15.The method of claim 14, wherein the CBD is an extract of Cannabis. 16.The method of claim 15, wherein the extract comprises less than 0.15%THC.
 17. The method of claim 9, further comprising treating a movementdisorder.
 18. The method of claim 9, further comprising treatingcognitive dysfunction.
 19. The method of claim 9, further comprisingtreating seizures.